中国药物警戒 ›› 2024, Vol. 21 ›› Issue (1): 89-93.
DOI: 10.19803/j.1672-8629.20230498

• 基础与临床研究 • 上一篇    下一篇

DMB衍生-高效液相色谱法测定重组人促卵泡激素中的唾液酸含量

王绿音, 李懿, 孙悦, 胡馨月, 张慧, 李晶#, 梁成罡*   

  1. 中国食品药品检定研究院化学药品检定所,国家药品监督管理局生物制品质量研究与评价重点实验室,国家药品监督管理局化学药品质量研究与评价重点实验室,北京 102629
  • 收稿日期:2023-08-16 出版日期:2024-01-15 发布日期:2024-01-18
  • 通讯作者: *梁成罡,男,研究员,激素类药物质量控制研究。E-mail: liangchenggang@nifdc.org.cn; # 为共同通信作者。
  • 作者简介:王绿音,女,硕士,助理研究员,激素类药物质量控制研究。
  • 基金资助:
    国家重点研发计划（2021YFF0600804）

Determination of sialic acid in recombinant human follicle stimulating hormone by high performance liquid chromatography with DMB derivatization

WANG Lyuyin, LI Yi, SUN Yue, HU Xinyue, ZHANG Hui, LI Jing, LIANG Chenggang*   

  1. Institute for Chemical Drug Control, National Institutes for Food and Drug Control, Key Laboratory of the Ministry of Health for Research on Quality Study and Evaluation of Biotech Products, Key Laboratory of the Ministry of Health for Research on Quality Study and Evaluation of Chemical Drugs, Beijing 102629, China
  • Received:2023-08-16 Online:2024-01-15 Published:2024-01-18

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摘要： 目的 建立采用 4,5-亚甲二氧基-1,2-苯二胺二盐酸盐（DMB）衍生-高效液相色谱-荧光检测器（HPLC - FLD）法（DMB衍生法）同时测定重组人促卵泡激素（rhFSH）中2种唾液酸组分N-羟乙酰神经氨酸（Neu5Gc）和N-乙酰神经氨酸（Neu5Ac）含量的方法。方法 将DMB衍生法与邻苯二胺衍生-高效液相色谱法（邻苯二胺衍生法）进行对比研究,对DMB衍生法中的水解条件进行优化并确定最终方法为rhFSH原液经酸水解后,采用DMB进行衍生,经Agilent ZORBAX 300SB-C18色谱柱（250 mm×4.6 mm,5 μm）分离,采用荧光检测器检测。结果 与邻苯二胺衍生法相比,DMB衍生法能够实现Neu5Gc与Neu5Ac相关杂质的有效分离。该方法Neu5Gc和Neu5Ac 分别在0.5 ~ 5.0 μg·mL-1（r=0.999 9）和25~ 250 μg·mL-1（r=0.999 8）浓度范围内与峰面积呈良好的线性关系;Neu5Gc 和Neu5Ac的检测限分别为11.07、10.30 ng·mL-1，定量限分别为33.21、20.60 ng·mL-1，两者回收率分别为（99.63±1.33）%（n=9）和（100.62±1.58）%（n=9），RSD分别为1.3%（n=9）和1.5%（n=9）。结论 该方法专属性强、灵敏度高、精密度和准确度良好，可用于准确测定rhFSH中唾液酸Neu5Gc和Neu5Ac的含量。

关键词: 重组人促卵泡激素, 高效液相色谱法, DMB衍生, 荧光检测器, N-羟乙酰神经氨酸, N-乙酰神经氨酸, 含量, 唾液酸

Abstract: Objective To develop a 4,5-methylenedioxy-1,2-phenylenediamine dihydrochloride (DMB) derivatization method based on high-performance liquid chromatography-fluorescence detector (HPLC-FLD)(DMB derivatization method) that can concurrently quantify both N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac) in rhFSH. Methods A comparative study was undertaken between the o-phenylenediamine derivatization method based on high-performance liquid chromatography(o-phenylenediamine derivatization method) and DMB derivatization method. The optimized DMB derivatization method with refined hydrolysis conditions was established and demonstrated as follows: The rhFSH solution was first subjected to acid hydrolysis, followed by derivatization with DMB. The resulting solution was then separated using an Agilent ZORBAX 300SB-C18 chromatographic column (250 mm×4.6 mm, 5 μm) and analyzed by a fluorescence detector. Results When compared to the o-phenylenediamine derivatization, the optimized DMB derivatization method proves to be more effective in separating Neu5Gc and Neu5Ac related impurities. The optimized DMB derivatization method showcases superior specificity, with both Neu5Gc and Neu5Ac demonstrating a strong linear relationship with peak area within the concentration ranges of 0.5~5.0 μg·mL-1(r=0.999 9) and 25~250 μg·mL-1 (r=0.999 8). The limit of detection and quantification (LOD and LOQ) for Neu5Gc are impressively low as 11.07 ng·mL-1and 33.21 ng·mL-1 respectively, comparable to the levels of Neu5Ac (LOD: 10.30 ng·mL-1, LOQ: 20.60 ng·mL-1). Besides, the recovery rates for Neu5Gc and Neu5Ac are (99.63±1.33)% (n=9) and (100.62±1.58)% (n=9), with low RSDs of 1.3% (n=9) and 1.5% (n=9), respectively. Conclusion The method demonstrates commendable specificity, exceptional sensitivity, and noteworthy precision and accuracy, which would be ideally suited for the precise quantification of Neu5Gc and Neu5Ac content in rhFSH.

Key words: recombinant human follicle stimulating hormone(rhFSH), high-performance liquid chromatography, DMB derivatization, fluorescence detector, N-glycolylneuraminic acid, N-acetylneuraminic acid, content, sialic acid

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